The present invention relates generally to the treatment of diseases, and more specifically, to the preparation and use of complexes containing receptor-binding internalized ligands NABD and cytocide-encoding agents to alter the function, gene expression, or viability of a cell in a therapeutic manner.
A major goal of treatment of neoplastic diseases and hyperproliferative disorders is to ablate the abnormally growing cells while leaving normal cells untouched. Various methods are under development for providing treatment, but none provide the requisite degree of specificity.
One method of treatment is to provide toxins. Immunotoxins and cytotoxins are protein conjugates of toxin molecules with either antibodies or factors which bind to receptors on target cells. Three major problems may limit the usefulness of immunotoxins. First. the antibodies may react with more than one cell surface molecule, thereby effecting delivery to multiple cell types, possibly including normal cells. Second, even if the antibody is specific, the antibody reactive molecule may be present on normal cells. Third, the toxin molecule may be toxic to cells prior to delivery and internalization. Cytotoxins suffer from similar disadvantages of specificity and toxicity. Another limitation in the therapeutic use of immunotoxins and cytotoxins is the relatively low ratio of therapeutic to toxic dosage. Additionally, it may be difficult to direct sufficient concentrations of the toxin into the cytoplasm and intracellular compartments in which the agent can exert its desired activity.
Given these limitations, cytotoxic therapy has been attempted using viral vectors to deliver DNA encoding the toxins into cells. If eukaryotic viruses are used, such as the retroviruses currently in use, they may recombine with host DNA to produce infectious virus. Moreover, because retroviral vectors are often inactivated by the complement system, use in vivo is limited. Retroviral vectors also lack specificity in delivery; receptors for most viral vectors are present on a large fraction, if not all, cells. Thus, infection with such a viral vector will infect normal as well as abnormal cells. Because of this general infection mechanism, it is not desirable for the viral vector to directly encode a cytotoxic molecule.
While delivery of nucleic acids offers advantages over delivery of cytotoxic proteins such as reduced toxicity prior to internalization, there is a need for high specificity of delivery, which is currently unavailable with the present systems.
In view of the problems associated with gene therapy, there is a compelling need for improved treatments which are more effective and are not associated with such disadvantages. The present invention exploits the use of conjugates which have increased specificity and deliver higher amounts of nucleic acids to targeted cells, while providing other related advantages.
The present invention generally provides therapeutic compositions. In one aspect, the composition has the formula: receptor-binding internalized ligandxe2x80x94nucleic acid binding domainxe2x80x94cytocide-encoding agent. The receptor-bindinginternalized ligand is a polypeptide reactive with a cell surface receptor, the nucleic acid binding domain binds to a nucleic acid. the cytocide-encoding agent is a nucleic acid molecule encoding a cytocide and which binds to the nucleic acid binding domain, and the composition binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor. In another aspect, the composition has the formula: receptor-binding internalized ligand-nucleic acid binding domain-prodrug-encoding agent.
In certain embodiments, the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor, VEGF receptor, HBEGF receptor, or a cytokine. In other embodiments, the cytocide-encoding agent encodes a protein that inhibits protein synthesis and is preferably a ribosome inactivating protein, most preferably saporin. The protein is gelonin or diphtheria toxin in other embodiments. In other embodiments, the prodrug-encoding agent encodes HSV-thymidine kinase.
The nucleic acid binding domain is poly-L-lysine in one embodiment. In other embodiments, the nucleic acid binding domain is a transcription factor selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and xcex2-sheet motif proteins. In other embodiments, the nucleic acid binding domain binds nonspecifically to nucleic acids and is selected from the group consisting of poly-L-lysine, protamine, histone and spermine. In a preferred embodiment, the nucleic acid binding domain binds the coding region of a ribosome inactivating protein such as saporin. In another preferred embodiment, FGF is conjugated to poly-L-lysine.
In yet other embodiments, the cytocide-encoding agent contains a tissue-specific promoter, such as alpha-crystalline, gamma-crystalline, xcex1-fetoprotein, CEA, prostate-specific antigen, erbB-2, tyrosinase, xcex1-actin, c-myc, VEGF receptor, FGF receptor or cyclin D.
In another aspect, the composition also contains a linker. In various embodiments, the linker increases the flexibility of the conjugate and is (GlymSerp)n, (Ala Ala Pro Ala)n, wherein n is 1 to 6, m is 1 to 6 and p is 1 to 4, or the linker is a disulfide bond.
In yet another aspect, the composition has the formula: receptor-binding internalized ligand-cytocide encoding agent-nucleic acid binding domain, wherein the receptor-binding internalized ligand is conjugated to the cytocide-encoding agent, which is bound to the nucleic acid binding domain to form a complex.
In other aspects, the invention provides methods for preventing excessive cell proliferation in the anterior eye following surgery, treating corneal clouding following excimer laser surgery, preventing closure of a trabeculectomy, preventing pterygii recurrence, treating hyperproliferative diseases in the back of the eye, such as macular degeneration, diabetic retinopathy and proliferative virtreal retinopathy, treating smooth muscle cell hyperplasia after a wound healing response to a procedure, e.g., vein grafting, endarterectomies and arteriovenous shunts and treating cancer. In these aspects, an effective amount of the compositions described above are administered.